Browsing by Subject "Mast cell"

Lymph nodes are organs of efficiency. Once activated, they essentially function to optimize and accelerate the production of the adaptive immune response, which has the potential to determine survival of the host during an initial infection and protect against repeated infections, should specific and appropriate immunological memory be sufficiently induced. We now have an understanding of the fundamental structure of lymph nodes and many of the interactions that occur within them throughout this process. Yet, lymph nodes are dynamic and malleable organs and much remains to be investigated with regards to their responses to various types of challenges. In this work, we examined multiple inflammatory scenarios and sought to understand the complex ways that lymph nodes can be externally targeted to impact immunity. First, we outline a novel mechanism of cellular communication, where cytokine messages from the periphery are delivered to draining lymph nodes during inflammation. These signals are sent as particles, released by mast cells, and demonstrate the ability of the infected tissue to communicate to lymph nodes and shape their responses. Based on these interactions, we also explored the ability to therapeutically or prophylactically modulate lymph node function, using bioengineered particles based on mast cell granules, containing encapsulated cytokines. When we used these particles as a vaccine adjuvant, we were able to polarize adaptive immune responses, such as to promote a Th1 phenotype, or enhance a specific attribute of the immune response, such as the production of high avidity antibodies. We then explore three examples of lymph node-targeting pathogens: Salmonella typhimurium, Yersinia pestis and Dengue virus. Each of these pathogens has a well-characterized lifecycle including colonization of draining lymph node tissue. In the case of S. typhimurim, we report that the virulence this pathogen depends on a specific shut down of the chemotactic signals in the lymph node that are required to maintain appropriate cellular localization within it. Our results demonstrate that these architecture changes allow S. typhimurim to target the adaptive immune process in lymph nodes and contribute to its spread in vivo and lethality to the host. With Y. pestis, similar targeting of cellular trafficking pathways occurs through the modulation of chemokine expression. Y. pestis appears to use the host's cellular trafficking pathways to spread to lymph nodes in two distinct waves, first exploiting dendritic cell movement to lymph nodes and then enhancing monocyte chemoattractants to replicate within monocytes in draining lymph nodes. These processes also promote bacterial spread in vivo and we further demonstrate that blocking monocyte chemotaxis can prolong the host's survival. In the third example of pathogen challenge, we report for the first time that mast cells can contribute functionally to immunosurveillance for viral pathogen, here, promoting cellular trafficking of innate immune cells, including NK cells, and limiting the spread of virus to draining lymph nodes. For each of these three examples of lymph node targeting by microbial pathogens, we provide data that modulation of cellular trafficking to and within lymph nodes can drastically influence the nature of the adaptive immune response and, therefore, the appropriateness of that response for meeting a unique infectious challenge. Cumulatively this work highlights that a balance exists between host and pathogen-driven modulation of lymph nodes, a key aspect of which is movement of cells within and into this organ. Cytokine and chemokine pathways are an area of vulnerability for the host when faced with host-adapted pathogens, yet the lymph node's underlying plasticity and the observation that slight modulations can be beneficial or detrimental to immunity also suggests the targeting of these pathways with therapeutic intentions and during vaccine design.

The mammalian urinary bladder is a highly specialized organ that must be able to withstand considerable amounts of osmotic pressure at its mucosal surface, in addition to maintaining an impenetrable barrier against potential pathogens. The lower urinary tract's virtually inevitable exposure to external microbial pathogens warrants efficient tissue-specialized defenses to maintain sterility. The observation that the bladder can become chronically infected with uropathogenic E.coli (UPEC) in combination with clinical observations that antibody responses following bladder infections are not detectable, suggest defects in the formation of adaptive immunity and immunological memory. We have identified a broadly immunosuppressive transcriptional program specific to the bladder, but not the kidney, during infection of the urinary tract that is dependent on tissue-resident mast cells. This mast cell-dependent phenomenon involves localized production of IL-10 and results in suppressed humoral and cell-mediated responses and bacterial persistence. Therefore, in addition to the previously described role of mast cells orchestrating the early innate immune responses in the bladder during infection, they subsequently play a tissue-specific immunosuppressive role. These findings may explain the prevalent recurrence of bladder infections and suggest the bladder as a site exhibiting an intrinsic degree of mast cell-maintained immune privilege.

Interestingly, though the bladder is not capable of initiating an effective adaptive immune response during bladder infections, we have generated data showing that it was possible to circumvent the immune limitations of the bladder to provoke a strong adaptive and protective immune response by vaccinating against UPEC at an alternate mucosal site. We reasoned that by immunizing the nasal regions of mice with a vaccine formulation comprising of FimH adhesin, a highly conserved adhesive moiety of type 1 fimbriae expressed on UPEC, and an effective mucosal adjuvant we would evoke protective immunity against UPEC infections. We found that a FimH vaccine coupled with either a mast cell activating adjuvant c48/80 or CpG oligodeoxynucleotide, a TLR9 agonist, evoked high levels of FimH specific IgG antibody in the serum and IgA in the urine of immunized mice. We also observed that following UPEC challenge, these FimH/adjuvant immunized mice exhibited significantly reduced bacterial load in the bladders compared to mice challenged with just FimH. These studies reveal that immunization of nasal regions with a FimH vaccine is an effective strategy to overcome the limitation in adaptive immunity observed in the bladder.

The key components of innate immune defense to pathogens are various migratory as well as tissue resident innate immune cells, however, their interactions with pathogens as well as their immune-orchestrating roles are often poorly understood. While immune cells encounter pathogens at barrier sites and mount the first line of defense, pathogens are well adapted to bypass, inactivate and even exploit the functions of these cells. Better understanding of the interactions between pathogens and innate immune cells can teach us how pathogens avoid or exploit immune cells and how to overcome these mechanisms of pathogenesis by therapeutic interventions. In this work, we examined two scenarios of pathogen invasion and sought to understand the complex ways of external targeting of innate immunocytes that can either benefit the pathogen or the host.

First, we studied the migratory innate immunocytes in draining lymph nodes upon entry of Yersinia pestis via the skin and identified how this plague-causing bacterium coopted host cell death pathways of infiltrated mononuclear phagocytes. By employing time-lapse microscopy and flow cytometry, we demonstrated that within the confines of infected lymph nodes, bacteria-triggered necroptotic cell death resulted in the release of intracellular bacteria into the extracellular environment and attracted neighboring phagocytic cells, promoting their infection by these recently released bacteria. This expansion of bacteria-bearing immune cells which eventually migrate to secondary lymph nodes, enables large numbers of Y. pestis to disseminate from one node to the next via the lymphatic system. We show this mechanism of dissemination being essential for the transition of plague from a bubonic to septicemic stage and demonstrate immunotherapeutic potential of necroptosis inhibitors.

Next, we focused on mast cells, a resident innate immunocyte in the context of skin infection by Staphylococcus aureus. We showed that connective tissue mast cells promoted recruitment of neutrophils at the early stage and CD301b+ dendritic cells at the later stages of infection, which played critical roles in infection control and repair, respectively. We further demonstrated that exogenous activation of skin mast cells via a mast cell-specific G protein-coupled receptor controlled infection as well as enhanced mobilization of dendritic cells to draining lymph nodes in a mast-cell dependent manner and protected mice from re-infection. Therefore, selective activation of mast cells appears to orchestrate immunomodulation integrating both the innate and adaptive immune arms.

These studies reveal the yin and yang of innate immune cells in two very different infectious settings. They emphasize how different strategies to target these cells at the immune checkpoints can be beneficial for host-directed therapy against bacterial infections.

Urinary tract infections (UTIs) are the second most common type of infection identified in the clinical setting and disproportionately afflict women. UTIs most frequently manifest in the form of infection of the lower urinary tract, involving the bladder. Uropathogens, particularly uropathogenic E. coli, progressively colonize the urethra and ascend to the bladder, where they initiate cystitis. In some cases, infection further ascends through the ureters and reaches the kidneys, where it causes pyelonephritis. Infection of both the upper and lower urinary tract can have serious ramifications for the host, and this is in large part due not to infection itself but to host-directed responses to bacterial insults.

In this thesis, I will describe and discuss two distinct aspects of UTIs. In the first study, in vivo work in a mouse model of urinary tract infection revealed a novel role for mast cells, which are tissue-resident granulated innate immune cells, in directing the detachment and death of epithelial cells during cystitis, facilitating the clearance of bacteria from the bladder. An ex vivo porcine bladder infection model suggested a specific role for mast cell granules and the proteases contained therein, which was corroborated with in vitro experiments utlizing isolated mast cell granules and human epithelial cells to demonstrate granule-induced exfoliation and cell death. From this work, it is clear that mast cells play a highly targeted role in modulating urothelial integrity during bladder infection by mediating host-directed epithelial loss.

In the second study described in this dissertation, the synergistic roles of both pyelonephritis and vesico-ureteric reflux (VUR), a congenital urinary tract defect that results in the improper backflow of urine from the bladder to the kidney, in the development of reflux nephropathy, a fibrotic host response characterized by renal scar formation, were elucidated in a series of in vivo experiments. Specifically, the C3H mouse, which is naturally susceptible to VUR, was utilized to characterize the dynamics of kidney infection and the onset of reflux nephropathy. Renal scarring was dependent on the presence of sustained kidney infection and the accompanying inflammatory response due to VUR, while neither transient infection nor reflux alone were sufficient to provoke nephropathy. Thus, the development of reflux nephropathy is dependent upon the confluence of both infection and VUR.

This body of work reveals the double-edged sword of the host inflammatory response to urinary tract infection. In the bladder, mast cell activation and degranulation leads to granule-induced epithelial exfoliation and consequently a reduction in the bacterial burden in the bladder. However, the sustained inflammatory response that accompanies pyelonephritis in vesico-ureteric reflux-affected individuals results in significant damage to the kidney without any accompanying reduction in infection. These findings highlight the dueling roles of the host inflammatory response to infection in the upper and lower urinary tract and strongly suggest that differential clinical approaches to cystitis and pyelonephritis are necessary to promote an effective mast cell in the bladder in the former and facilitate the clearance of renal infection while mitigating tissue damage in the latter.

Mast cells, best known for their pathologic role in allergy, have recently been shown to have key roles in the initiation of adaptive immune responses. These cells are located throughout the body just beneath barriers separating host from environment, possess multiple pathogen recognition systems, and store large quantities of fully active inflammatory mediators. These key features make them uniquely situated to act as sentinels of immunity, releasing the very earliest alarm signals when a pathogen is present. As a testament to the importance of these cells, mast cell-deficient mice have suboptimal immune responses, and mast cell activators can act as potent adjuvants for experimental immunizations. Specifically, mast cells have been shown to enhance the number of naive lymphocytes in infection site-draining lymph nodes, and to encourage the migration of dendritic cells to responding lymph nodes.

Although infections usually occur at peripheral sites, adaptive immune responses are initiated in distant lymph nodes. Despite the distance, signals from the site of infection result in dramatic, rapid reorganization of the node, including massive recruitment of naive lymphocytes from the circulation and extensive vascular restructuring to accommodate the increase in size. How such signals reach the lymph node is not well understood.

When mast cells degranulate, in addition to releasing soluble mediators such as histamine, they expel large, stable, insoluble particles composed primarily of heparin and cationic proteins. The work presented herein demonstrates that these particles act as extracellular chaperones for inflammatory mediators, protecting them from dilution into the interstitial space, degradation, and interaction with non-target host cells and molecules. The data show clearly that mast cells release such particles, that they are highly stable, that they contain tumor necrosis factor (a critically important immunomodulator), and that they can traffic from peripheral sites to draining lymph nodes via lymphatic vessels. Furthermore, extensive biochemical characterization of purified mast cell-derived particles was performed. Finally, evidence is presented that such particles can elicit lymph node enlargement, an infection-associated phenomenon that favors the development of adaptive immunity, by delivering peripheral TNF to draining lymph nodes.

This signaling concept, that particles may chaperone signals between distant sites, also has important implications for adjuvant design. The evidence presented here shows that encapsulation of TNF into synthetic particles similar to mast cell-derived particles greatly enhances its potency for eliciting lymph node enlargement, an indication that adaptive immunity may be improved. This delivery system should ensure that more adjuvant arrives in the draining lymph node intact, where it would lead to changes favorable to the development of the immune response. Such a system would also facilitate the delivery of multi-component adjuvants that would act synergistically at the level of the lymph node when gradually released from microparticle carriers. An additional advantage of microparticle encapsulation is that vaccine formulations of this type may require much lower doses of expensive antigen and adjuvants.

The delivery of inflammatory mediators to lymph nodes during immune responses may be an important general feature of host defense. Although the action of mediators of peripheral origin on draining lymph nodes has been described before, this is the first demonstration of a specific adaptation to deliver such mediators. Not only is the characterization of mast cell-derived particles important to basic immunology, but mimicking this adaptation may also lead to improved therapeutics.

Mast cells (MCs) are increasingly recognized as powerful sentinel cells responsible for modulating the early immune responses to a wide range of infectious agents. This protective role is attributable in part to their preponderance at the host-environment interface and their innate capacity to rapidly release modulators of immune cell trafficking which promotes the early recruitment of pathogen-clearing immune cells from the blood. However, host-adapted pathogens had been a critical threat to human for a long time because they have evolved mechanisms directed at overcoming protective immunity.

In this work, we outline Salmonella enterica serovar Typhimurium has evolved a novel mechanism to inactivate peripheral MCs resulting in limited neutrophil responses at infection sites in early stage of infection. Because of the delay in bacterial clearance at the point of entry, Salmonella are able to multiply and rapidly disseminate to distal sites. Suppression of local MCs' degranulation restricted outflow of vascular contents into infection sites, thus facilitating bacterial spread.

We discover MC suppression is mediated by the Salmonella Protein Tyrosine Phosphatase (SptP), which shares structural homology with Yersinia YopH. Interestingly, SptP, not only shares homology with phosphatases found in MCs, they are also homologous to YopH an effector protein expressed by plague causing Yersinia pestis. We show that YopH had MC suppressing abilities as SptP suggesting that this activity is shared among some of the more virulent bacterial pathogens. The functionally relevant domain in SptP is its enzymatic site and that it works by dephosphorylating the vesicle fusion protein N-ethylmalemide-sensitive factor (NSF) and by blocking phosphorylation of Syk, which is located in downstream and upstream of tyrosine phosphorylation signaling pathway in MCs.

Without SptP, orally challenged S. Typhimurium failed to suppress MC degranulation and exhibited limited colonization of the mesenteric lymph nodes. Administration of SptP to sites of Escherichia coli infection markedly enhanced its virulence. Thus, SptP-mediated inactivation of local MCs is a powerful mechanism utilized by S. Typhimurium to impede early innate immunity. This finding provides a logical explanation for why previous attempts by others to demonstrate a protective role for MCs against Salmonella infections have resulted in equivocal results.

Taken together, this work highlights an overlooked virulence mechanism possessed by certain host adapted pathogens to avoid the host's innate immune system. Additionally, this innate immune-quelling property of SptP may hold future promise in tempering harmful inflammatory disorders in the body of an immune competent host.

IgE-mediated anaphylaxis is a dangerous systemic reaction to allergens and affect up to 50 million people in the United States, with the number of cases increasing every year. This reaction occurs when allergens enter the bloodstream and are recognized by IgE-sensitized mast cells (MCs), which leads to massive release of MC granules systemically. Mediators released from these granules have historically been believed to cause a sudden drop in blood pressure and core body temperature by promoting vasodilation and vascular leakage. However, here, we present evidence that in mice, the nervous system, specifically the thermoregulatory neural circuit, also plays a role in the drop in body temperature during IgE-mediated anaphylaxis. This neural circuit is activated by granule-borne chymase from mast cells and is sensed and encoded as a "pseudo-heat" signal, which leads to the activation of the warm thermoregulatory neural network. This activation then rapidly reduces thermogenesis in brown adipose tissue, contributing to hypothermia. Compared to wild-type mice, mice lacking chymase or TRPV1 (a receptor on sensory neurons) had a lesser drop in body temperature during IgE-mediated anaphylaxis. Additionally, systemic activation of TRPV1+ sensory neurons is sufficient to induce anaphylactic-like responses. Therefore, mast cell mediators, especially chymase, promote IgE-mediated anaphylaxis not only through their effects on blood vessels but also via the TRPV1-thermoregulatory neural circuit axis.